Title

Mutagenesis Studies to Investigate Ligand Binding to the Carbon Monoxide-Sensing Heme Protein, CooA, from Carboxydothermus Hydrogenoformans

Document Type

Article

Publication Date

4-2010

Abstract

The work reported here is part of a larger effort to determine how amino acid residues that compose the heme pockets of gas-sensing heme proteins impact the proteins’ regulation mechanisms and gas specificity. In the present study, we have employed site-directed mutagenesis to prepare protein variants of the carbon monoxide (CO)-sensing heme protein, CooA, from Carboxydothermus hydrogenoformans. These mutants have been designed in an attempt to rationally alter CooA’s effector specificity. We have prepared, isolated, and purified several protein variants to date, including C80T CooA. The C80T substitution is expected to increase the steric bulk on the proximal face of the CooA heme, but eliminates a potential site of S-nitrosylation that may impact protein activation by nitric oxide (NO). Currently, we are investigating both C80T heme structure and this variant’s ability to be activated for DNA-binding by CO and NO. Our next goal will be to prepare mutants that allow us to study proposed cooperative interactions that take place between the monomers of the CooA homodimer. The authors wish to thank the Valparaiso University College of Arts and Sciences for financial support.